A requirement for septins and the autophagy receptor p62 in the proliferation of intracellular Shigella.

Shigella flexneri, a Gram-negative enteroinvasive pathogen, causes inflammatory destruction of the human intestinal epithelium. During infection of epithelial cells, Shigella escape from the phagosome to the cytosol, where they reroute host cell glycolysis to obtain nutrients for proliferation. Septins, a poorly understood component of the cytoskeleton, can entrap cytosolic Shigella targeted to autophagy in cage-like structures to restrict bacterial proliferation. Although bacterial entrapment by septin caging has been the subject of intense investigation, the role of septins and the autophagy machinery in the proliferation of noncaged Shigella is mostly unknown. Here, we found that intracellular Shigella fail to efficiently proliferate in SEPT2-, SEPT7-, or p62/SQSTM1-depleted cells. Consistent with a failure to proliferate, single cell analysis of bacteria not entrapped in septin cages showed that the number of metabolically active Shigella in septin- or p62-depleted cells is reduced. Targeted metabolomic analysis revealed that host cell glycolysis is dysregulated in septin-depleted cells, suggesting a key role for septins in modulation of glycolysis. Together, these results suggest that septins and the autophagy machinery may regulate metabolic pathways that promote the proliferation of intracellular Shigella not entrapped in septin cages.

Mechanical force induces mitochondrial fission.

Eukaryotic cells are densely packed with macromolecular complexes and intertwining organelles, continually transported and reshaped. Intriguingly, organelles avoid clashing and entangling with each other in such limited space. Mitochondria form extensive networks constantly remodeled by fission and fusion. Here, we show that mitochondrial fission is triggered by mechanical forces. Mechano-stimulation of mitochondria - via encounter with motile intracellular pathogens, via external pressure applied by an atomic force microscope, or via cell migration across uneven microsurfaces - results in the recruitment of the mitochondrial fission machinery, and subsequent division. We propose that MFF, owing to affinity for narrow mitochondria, acts as a membrane-bound force sensor to recruit the fission machinery to mechanically strained sites. Thus, mitochondria adapt to the environment by sensing and responding to biomechanical cues. Our findings that mechanical triggers can be coupled to biochemical responses in membrane dynamics may explain how organelles orderly cohabit in the crowded cytoplasm.

Wiskott-Aldrich syndrome protein regulates autophagy and inflammasome activity in innate immune cells.

Dysregulation of autophagy and inflammasome activity contributes to the development of auto-inflammatory diseases. Emerging evidence highlights the importance of the actin cytoskeleton in modulating inflammatory responses. Here we show that deficiency of Wiskott-Aldrich syndrome protein (WASp), which signals to the actin cytoskeleton, modulates autophagy and inflammasome function. In a model of sterile inflammation utilizing TLR4 ligation followed by ATP or nigericin treatment, inflammasome activation is enhanced in monocytes from WAS patients and in WAS-knockout mouse dendritic cells. In ex vivo models of enteropathogenic Escherichia coli and Shigella flexneri infection, WASp deficiency causes defective bacterial clearance, excessive inflammasome activation and host cell death that are associated with dysregulated septin cage-like formation, impaired autophagic p62/LC3 recruitment and defective formation of canonical autophagosomes. Taken together, we propose that dysregulation of autophagy and inflammasome activities contribute to the autoinflammatory manifestations of WAS, thereby identifying potential targets for therapeutic intervention.

Mitochondria mediate septin cage assembly to promote autophagy of Shigella.

Septins, cytoskeletal proteins with well-characterised roles in cytokinesis, form cage-like structures around cytosolic Shigella flexneri and promote their targeting to autophagosomes. However, the processes underlying septin cage assembly, and whether they influence S. flexneri proliferation, remain to be established. Using single-cell analysis, we show that the septin cages inhibit S. flexneri proliferation. To study mechanisms of septin cage assembly, we used proteomics and found mitochondrial proteins associate with septins in S. flexneri-infected cells. Strikingly, mitochondria associated with S. flexneri promote septin assembly into cages that entrap bacteria for autophagy. We demonstrate that the cytosolic GTPase dynamin-related protein 1 (Drp1) interacts with septins to enhance mitochondrial fission. To avoid autophagy, actin-polymerising Shigella fragment mitochondria to escape from septin caging. Our results demonstrate a role for mitochondria in anti-Shigella autophagy and uncover a fundamental link between septin assembly and mitochondria.

The zebrafish as a new model for the in vivo study of Shigella flexneri interaction with phagocytes and bacterial autophagy.

Autophagy, an ancient and highly conserved intracellular degradation process, is viewed as a critical component of innate immunity because of its ability to deliver cytosolic bacteria to the lysosome. However, the role of bacterial autophagy in vivo remains poorly understood. The zebrafish (Danio rerio) has emerged as a vertebrate model for the study of infections because it is optically accessible at the larval stages when the innate immune system is already functional. Here, we have characterized the susceptibility of zebrafish larvae to Shigella flexneri, a paradigm for bacterial autophagy, and have used this model to study Shigella-phagocyte interactions in vivo. Depending on the dose, S. flexneri injected in zebrafish larvae were either cleared in a few days or resulted in a progressive and ultimately fatal infection. Using high resolution live imaging, we found that S. flexneri were rapidly engulfed by macrophages and neutrophils; moreover we discovered a scavenger role for neutrophils in eliminating infected dead macrophages and non-immune cell types that failed to control Shigella infection. We observed that intracellular S. flexneri could escape to the cytosol, induce septin caging and be targeted to autophagy in vivo. Depletion of p62 (sequestosome 1 or SQSTM1), an adaptor protein critical for bacterial autophagy in vitro, significantly increased bacterial burden and host susceptibility to infection. These results show the zebrafish larva as a new model for the study of S. flexneri interaction with phagocytes, and the manipulation of autophagy for anti-bacterial therapy in vivo.

The secretome of Helicobacter trogontum.

BACKGROUND: Helicobacter trogontum is a putative enterohepatic pathogen, which following infection of IL-10 knock-out mice, results in severe clinical signs and typhlocolitis. MATERIALS AND METHODS: The pathogenic potential of H. trogontum Type strain LRB 8581 was investigated using proteomics coupled with mass spectrometry to characterize the secretome of H. trogontum and scanning electron microscopy to visualize H. trogontum adherence and invasion. RESULTS: One hundred and four proteins were identified and bioinformatically predicted to be secreted. Further functional classifications revealed proteins involved in motility, virulence, and colonization factors and the type VI secretion system. Microscopy showed that H. trogontum can adhere to host cells through flagella-microvillus interactions and invade causing a membrane ruffling-like effect and severe cell damage. CONCLUSIONS: This indicated H. trogontum has the ability to adhere to and invade human cells and secrete factors that may contribute to disease development.

The pathogenic potential of Helicobacter pullorum: possible role for the type VI secretion system.

BACKGROUND: Helicobacter pullorum is a putative enterohepatic pathogen that has been associated with hepatobiliary and gastrointestinal diseases in chickens and in humans. The pathogenic potential of H. pullorum NCTC 12826 was investigated. METHODS: Adherence and gentamicin protection assays and scanning electron microscopy were performed to quantitate and visualise H. pullorum adherence and invasion. Proteomics coupled with mass spectrometry was employed to characterise the secretome of H. pullorum. RESULTS: Helicobacter pullorum was able to adhere to the Caco-2 intestinal epithelial cell line with a mean attachment value of 1.98 ± 0.16% and invade Caco-2 cells with a mean invasion value of 0.25 ± 0.02%. The in vitro adherence and invasion assays were confirmed with scanning electron microscopy, which showed that H. pullorum can adhere to host cells through flagellum-microvillus interaction and invade causing a membrane-ruffling effect. One hundred and thirty-seven proteins were identified, of which 33 were bioinformatically predicted to be secreted. Further functional classifications revealed six putative virulence and colonisation factors, which included cell-binding factor 2, flagellin, secreted protein Hcp, valine-glycine repeat protein G, a type VI secretion protein, and a protease. Protein threading of H. pullorum Hcp and subsequent 3D-Blast searches revealed structural similarities between Hcp and endocytic vesicle coat proteins, suggesting the type VI secretion system of H. pullorum may interact with endocytic vesicles. CONCLUSIONS: This study has shown that H. pullorum has the ability to adhere to and invade human cells and secrete factors that may contribute to the pathogenic potential of H. pullorum.